Characteristics of the antimicrobial resistance of Staphylococcus aureus isolated from chicken meat produced by different integrated broiler operations in Korea

Characteristics of the antimicrobial resistance of Staphylococcus aureus isolated from chicken... Abstract Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The purpose of this study was to investigate the prevalence of Staphylococcus aureus (S. aureus) and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems in Korea. Among 200 chicken meat samples, 94 were observed to be positive for S. aureus. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among the operations. Four methicillin-resistant S. aureus isolates (MRSA) were recovered from 3 different operations. A high proportion of the S. aureus isolates were resistant to penicillins (51.2%), tetracycline (38.8%), and ciprofloxacin (CIP; 33.9%). Especially, 3 different operations showed a high number of CIP resistance (45.5∼100%) and multidrug resistance (50.0∼100%). Among 41 CIP-resistant S. aureus isolates, 75.6% showed a double amino-acid exchange of both gyrA and parC, with CIP minimum inhibitory concentrations (MIC) of ≥32 μg/mL. Four MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, exhibiting oxacillin, CIP, and enrofloxacin MIC ranges of 16 to 128, 32 to 64, and 8 to 128 μg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Our findings suggest that S. aureus with resistance to important antimicrobial compounds can now be found in association with integrated broiler operations, providing the data to support the development of a monitoring and prevention program in integrated operations. INTRODUCTION Staphylococcus aureus (S. aureus) is one of the most common causes of foodborne diseases in the world and produces gastrointestinal illness through a wide variety of toxins, including staphylococcal enterotoxins. In Korea, S. aureus was the fourth most frequent pathogen, after pathogenic Escherichia coli, Salmonella, and Vibrio spp. (Hyeon et al., 2013). The Korea Food and Drug Administration reported that a total of 1,472 cases were reported from 38 staphylococcal food poisoning outbreaks from 2012 to 2016 in Korea. (http://foodsafetykorea.go.kr/portal/healthyfoodlife/foodPoisoningStat.do?menu_grp=MENU_NEW02&menu_no=2786). S. aureus is a common commensal of the skin and mucosal membranes of humans, with estimates of 20 to 30% for persistent and 60% for intermittent colonizations (Kluytmans and Wertheim, 2005). However, S. aureus of animal origin may be transmitted to humans through direct contact with animals or via exposure to contaminated food (Lowder et al., 2009). Epidemiologically, poultry meat is of paramount importance and is still inculpated as a primary source of human food poisoning (Abdalrahman et al., 2015; Owuna et al., 2015; Bortolaia et al., 2016). Zoonotic transmission of S. aureus from poultry has usually been studied in relation to antimicrobial resistance. It is considered to be a significantly disturbing issue in the poultry industry owing to its impact on public health, and is a challenge to medical and veterinary officials worldwide (APUA, 2010; Ruban and Fairoze, 2011). Especially, methicillin-resistant S. aureus (MRSA) has been isolated in chicken meats (Lim et at., 2010; Velasco et al., 2015) and is considered a source of human infections caused by consuming contaminated food products made from animals (Lee, 2003). The Korea Animal Health Products Association reported that around 1,500 tons of antimicrobials were sold each year during 2003 to 2007, but less than 1,000 tons were sold for the 4 consecutive years from 2011 to 2015. Although tetracyclines and penicillins were the largest selling antimicrobials, the sales of most antimicrobials have gradually decreased. However, the sales of phenicols and cephalosporins have increased by 3 and 5 times from 2007 to 2015, respectively (QIA, 2015). Presently, the broiler chicken industry operates largely through vertical integration, with company ownership of breeding farms, multiplication farms, hatcheries, feed mills, some broiler growing farms, and processing plants. In Korea, several large integrated companies supply about 80% of the broiler chickens on the market, (KAPE, 2015). The livestock on the integrated farms, which includes chicken, is reared intensively, and antimicrobial agents are used as growth promoters and for prophylactic and therapeutic treatments. The objective of this study was to investigate the prevalence of S. aureus and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems. MATERIALS AND METHODS Sample collection A total of 200 chicken meats was collected at 4 retail markets, visiting 3 or more times during the year 2016. These meats were produced by 50 broiler farms and divided into 7 different integrated broiler operations supplying about 80% of the broiler chickens in Korea. Four meats from the same farm origin were sampled and tested for this study. Each meat was aseptically placed into a vacuum bag (Sealed Air, Elmwood Park, NJ), and 400mL of sterile buffered peptone water (BD Biosciences, Sparks, MD) were added. The bag was shaken 50 times, and approximately 50mL of the rinse water were transferred into a sterile specimen cup. Bacterial isolation Microbiological analyses were performed according to the Processing and Ingredients Specification of Livestock Products published by the Ministry of Food and Drug Safety (KFDS, 2014). In brief, 25 mL of meat rinse water were added to 225 mL of tryptic soy broth (TSB; BD Biosciences) containing 10% NaCl and incubated at 37°C for 20 to 24 hours. After enrichment, the bacteria-containing TSB was streaked onto Baird-Parker agar (Oxoid Ltd., Hampshire, UK) with egg yolk tellurite emulsion (Oxoid) and incubated at 37°C for 48 hours. Two typical colonies of S. aureus from one meat origin were picked and identified by standard microbiological culture-based tests, including Gram staining; catalase (using 3% hydrogen peroxide), indole, oxidase, coagulase, citrate, urease, Voges-Proskauer, and DNase testing; and tests for sugar fermentation and mannitol salt agar oxidation and fermentation (Cruickshank et al., 1975). PCR identification of S. aureus was carried out as described by Manson et al. (2001). If 2 isolates from the same origin showed the same antimicrobial susceptibility patterns, then only one isolate was randomly chosen and included in this study. Antimicrobial susceptibility tests All S. aureus isolates were investigated for their antimicrobial resistance with the disc diffusion test, using the following antibiotic discs (BD): amikacin (AN, 30 μg), amoxicillin/clavulanic acid (AmC, 20/10 μg), ampicillin (10 μg), cefazolin (30 μg), cefepime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CC, 2 μg), doxycycline (DOX, 30 μg), erythromycin (E, 15 μg), gentamicin (G, 10 μg), kanamycin (30 μg), penicillin (P, 10 μg), rifampin (5 μg), sulfamethoxazole/trimethoprim (23.75/1.25 μg), tetracycline (TE, 30 μg) and vancomycin (VA, 30 μg), and nitrofurantoin (F/M, 300 μg) and teicoplanin (TEC, 30 μg) purchased from Oxoid. Results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2013). Multidrug resistance (MDR) was defined as acquired non-susceptibility to at least one agent in 3 or more antimicrobial categories. Minimum inhibitory concentrations The minimum inhibitory concentrations (MIC) were determined by standard agar dilution methods with Mueller–Hinton agar (BD Biosciences), according to the recommendations of the CLSI (2013). The antimicrobial agents used in this study were CIP, enrofloxacin (ENR), and oxacillin (OX) at concentrations ranging from 0.125 to 256 μg/mL. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The breakpoint was determined using the S. aureus criteria set by the CLSI (2013). S. aureus ATCC 29,213 was used as a quality control, as recommended by the CLSI. Screening for mutations in gyrA and parC For CIP-resistant strains, PCR amplification of the quinolone resistance-determining region (QRDR) of gyrA and parC was carried out using the primers: gyrA-F (5΄-AATGAACAAGGTATGACACC-3΄) and gyrA-R (5΄-TACGCGCTTCAGTATAACGC-3΄); and parC-F (5΄-ACTTGAAGATGTTTTAGGTGAT-3΄) and parC-R (5΄-TTAGGAAATCTTGATGGCAA-3΄) (Kwak et al., 2013). The amplified DNA was purified using a PCR purification kit (Qiagen, Valencia, CA) and sequenced (Cosmogen Tech, Daejoen, Korea). The DNA sequences were compared with those of the standard gyrA (GenBank Accession No. ABD29197.1) and parC (GenBank Accession No. ABD30448.1) genes. Detection of mecA and pvl The mecA and pvl genes were detected by PCR using the primers as described previously (Lina et al., 1999; Oliveira and Lencastre, 2002) RESULTS Prevalence of S. aureus The prevalence of S. aureus in the chicken meats collected from a retail market is shown in Table 1. Among 200 chicken meats, 94 were observed to be positive for S. aureus. Among chicken meats that had originated from 7 integrated broiler chicken operations, those from operation C had the highest prevalence of S. aureus (59.4%, 19 of 32 samples), and operation E showed the lowest prevalence (25.0%, 3 of 12 samples). Four MRSA isolates were obtained from operations B (one isolate), C (2 isolates), and E (one isolate). However, a higher number of CIP-resistant S. aureus isolates were obtained from operations B (45.5%, 15 of 33 isolates), D (66.7%, 8 of 12 isolates), and G (100%, 5 of 5 isolates). Table 1. Prevalence of Staphylococcus aureus from 7 integrated broiler chicken operations.   Integrated broiler chicken operations  A  B  C  D  E  F  G  Total (%)  No. of farms  14  11  8  8  3  3  3  50  No. of chicken meat tested  56  44  32  32  12  12  12  200  No. of chicken meat isolated Staphylococcus aureus (%)  28 (53.8)  22 (50.0)  19 (59.4)  11 (34.4)  3 (25.0)  7 (58.3)  4 (33.3)  94 (47.0)  No. of Staphylococcus aureus isolatesa  32  33  28  12  3  8  5  121  No. of Methicillin- resistant Staphylococcus aureus (%)  0 (0.0)  1 (3.0)  2 (7.1)  0 (0.0)  1(33.3)  0 (0.0)  0 (0.0)  4 (3.3)  No. of Ciprofloxacin- resistant Staphylococcus aureus (%)  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)    Integrated broiler chicken operations  A  B  C  D  E  F  G  Total (%)  No. of farms  14  11  8  8  3  3  3  50  No. of chicken meat tested  56  44  32  32  12  12  12  200  No. of chicken meat isolated Staphylococcus aureus (%)  28 (53.8)  22 (50.0)  19 (59.4)  11 (34.4)  3 (25.0)  7 (58.3)  4 (33.3)  94 (47.0)  No. of Staphylococcus aureus isolatesa  32  33  28  12  3  8  5  121  No. of Methicillin- resistant Staphylococcus aureus (%)  0 (0.0)  1 (3.0)  2 (7.1)  0 (0.0)  1(33.3)  0 (0.0)  0 (0.0)  4 (3.3)  No. of Ciprofloxacin- resistant Staphylococcus aureus (%)  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  aIf two isolates from the same origin showed the same antimicrobial susceptibility patterns, only one isolate was included. View Large Antimicrobial resistance profile The results of the antimicrobial resistance analysis of the S. aureus isolates are shown in Table 2. A high proportion of S. aureus isolates was resistant to penicillins (62 isolates, 51.2%), TE (47 isolates, 38.8%), CIP (41 isolates, 33.9%), K (30 isolates, 24.7%), DOX (25 isolates, 20.7%), and E (25 isolates, 20.7%). Although isolates from operations C and F showed only 25.0 and 12.5% resistance to penicillins, respectively, the isolates from the other integrated operations were the most frequently resistant to this antibiotic. Moreover, isolates from operation B showed the highest resistance to K (48.5%) and TE (48.5%), those from operation D had the highest resistance to TE (58.3%) and CIP (66.7%), and isolates from operation G were the most resistant to AmC (60.0%), TE (60.0%), DOX (60.0%), and CIP (100%). Table 2. Distribution of antimicrobial resistance of 121 Staphylococcus aureus from 7 integrated broiler chicken operations.   Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  PNICILLINS  Penicillin  25 (78.1)a  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  Ampicillin  25 (78.1)  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS  Amoxicillin/Clavulanic acid  1 (3.1)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  3 (60.0)  6 (5.0)  CEPHEMS (PARENTERAL)  Cefazolin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cephalothin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefuroxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefotaxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Ceftazidime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  1 (20.0)  3 (2.5)  Cefepime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  AMIOGLYCOSIDES  Gentamicin  1 (3.1)  12 (36.4)  8 (28.6)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  23 (19.0)  Kanamycin  2 (6.3)  16 (48.5)  10 (35.7)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  30 (24.7)  MARCROLIDES  Erythromycin  6 (18.8)  9 (27.3)  4 (14.3)  4 (33.3)  1 (33.3)  1 (12.5)  0 (0.0)  25 (20.7)  TETEACYCLINES  Tetracycline  7 (21.9)  16 (48.5)  12 (42.9)  7 (58.3)  1 (33.3)  1 (12.5)  3 (60.0)  47 (38.8)  Doxycycline  6 (18.8)  3 (9.1)  8 (28.6)  3 (25.0)  1 (33.3)  1 (12.5)  3 (60.0)  25 (20.7)  FLUOROQUINOLONES  Ciprofloxacin  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  LINCOSAMIDES  Clindamycin  6 (18.8)  9 (27.3)  3 (10.7)  4 (33.3)  1 (33.3)  0 (0.0)  0 (0.0)  23 (19.0)  FOLATE PATHWAY INHIBITORS  Sulfamethoxazole/Trimethoprim  1 (3.1)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (0.8)  PHENICOLS  Chloramphenicol  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (12.5)  2 (40.0)  3 (2.5)  ANSAMYCINS  Rifampin  0 (0.0)  0 (0.0)  1 (3.5)  0 (0.0)  0 (0.0)  1 (12.5)  1 (20.0)  3 (2.5)    Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  PNICILLINS  Penicillin  25 (78.1)a  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  Ampicillin  25 (78.1)  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS  Amoxicillin/Clavulanic acid  1 (3.1)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  3 (60.0)  6 (5.0)  CEPHEMS (PARENTERAL)  Cefazolin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cephalothin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefuroxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefotaxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Ceftazidime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  1 (20.0)  3 (2.5)  Cefepime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  AMIOGLYCOSIDES  Gentamicin  1 (3.1)  12 (36.4)  8 (28.6)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  23 (19.0)  Kanamycin  2 (6.3)  16 (48.5)  10 (35.7)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  30 (24.7)  MARCROLIDES  Erythromycin  6 (18.8)  9 (27.3)  4 (14.3)  4 (33.3)  1 (33.3)  1 (12.5)  0 (0.0)  25 (20.7)  TETEACYCLINES  Tetracycline  7 (21.9)  16 (48.5)  12 (42.9)  7 (58.3)  1 (33.3)  1 (12.5)  3 (60.0)  47 (38.8)  Doxycycline  6 (18.8)  3 (9.1)  8 (28.6)  3 (25.0)  1 (33.3)  1 (12.5)  3 (60.0)  25 (20.7)  FLUOROQUINOLONES  Ciprofloxacin  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  LINCOSAMIDES  Clindamycin  6 (18.8)  9 (27.3)  3 (10.7)  4 (33.3)  1 (33.3)  0 (0.0)  0 (0.0)  23 (19.0)  FOLATE PATHWAY INHIBITORS  Sulfamethoxazole/Trimethoprim  1 (3.1)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (0.8)  PHENICOLS  Chloramphenicol  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (12.5)  2 (40.0)  3 (2.5)  ANSAMYCINS  Rifampin  0 (0.0)  0 (0.0)  1 (3.5)  0 (0.0)  0 (0.0)  1 (12.5)  1 (20.0)  3 (2.5)  aNo. of isolates shown resistance (%). All isolates showed sensitivity to kanamycin, nitrofurantoin, vancomycin, and teicoplanin tested in this study. View Large The distribution of MDR is shown in Table 3. Forty-three of 121 isolates showed resistance from 3 to 7 classes of antimicrobial agents. Operations B (51.5%, 17 of 33 isolates), D (50.0%, 6 of 12 isolates), and G (100%, 5 of 5 isolates), in particular, showed a high proportion of isolates with MDR. Table 3. Multidrug resistance patterns among 121 Staphylococcus aureus from 7 integrated broiler chicken operations. Antimicrobial resistance patterns  No. of antimicrobials  No. of classes  Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  AM·AmC·CAZ·CC·CF·CIP·  14  7          1 (33.3)      1 (0.8)  CTX·CXM·CZ·DOX·E·FEP·P·TE                      AM·AmC·CAZ·CC·CF·CIP·  13  7    1 (3.0)            1 (0.8)  CTX·CXM·CZ·E·FEP·P·TE                      AM·C·CC·CIP·E·P·K·SXT  8  7  1 (3.1)              1 (0.8)  AM·C·CC·CIP·CTX·E·P·TE  8  6  2 (6.3)              2 (1.7)  AM·CC·CIP·DOX·E·P·TE  7  5  3 (9.4)  2 (6.1)            5 (4.1)  AM·CC·E·G·K·P·TE  7  5    1 (3.0)            1 (0.8)  AM·CC·CIP·E·P·TE  6  5    1 (3.0)  3 (10.7)  2 (16.7)        6 (5.0)  AM·AmC·CIP·DOX·P·TE  6  4              3 (60.0)  3 (2.5)  AM·CC·CIP·E·P  5  4    2 (6.1)    2 (16.7)        4 (3.3)  C·CIP·G·K·RA  5  4              1 (20.0)  1 (0.8)  C·DOX·E·RA·TE  5  4            1 (12.5)    1 (0.8)  CAZ·CC·CIP·G·K  5  4              1 (20.0)  1 (0.8)  AM·CIP·DOX·P·TE  5  3  1 (3.1)      1 (8.3)        2 (1.7)  AM·G·K·P·TE  5  3    6 (18.2)            6 (5.0)  AM·CIP·DOX·P  4  3      1 (3.5)          1 (0.8)  AM·CIP·P·TE  4  3  1 (3.1)    1 (3.5)  1 (8.3)        3 (2.5)  AM·K·P·TE  4  3    4 (12.1)            4 (3.3)  Total (%)      8 (25.0)  17 (51.5)  5 (17.9)  6 (50.0)  1 (33.3)  1 (12.5)  5 (100.0)  43 (35.5)  Antimicrobial resistance patterns  No. of antimicrobials  No. of classes  Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  AM·AmC·CAZ·CC·CF·CIP·  14  7          1 (33.3)      1 (0.8)  CTX·CXM·CZ·DOX·E·FEP·P·TE                      AM·AmC·CAZ·CC·CF·CIP·  13  7    1 (3.0)            1 (0.8)  CTX·CXM·CZ·E·FEP·P·TE                      AM·C·CC·CIP·E·P·K·SXT  8  7  1 (3.1)              1 (0.8)  AM·C·CC·CIP·CTX·E·P·TE  8  6  2 (6.3)              2 (1.7)  AM·CC·CIP·DOX·E·P·TE  7  5  3 (9.4)  2 (6.1)            5 (4.1)  AM·CC·E·G·K·P·TE  7  5    1 (3.0)            1 (0.8)  AM·CC·CIP·E·P·TE  6  5    1 (3.0)  3 (10.7)  2 (16.7)        6 (5.0)  AM·AmC·CIP·DOX·P·TE  6  4              3 (60.0)  3 (2.5)  AM·CC·CIP·E·P  5  4    2 (6.1)    2 (16.7)        4 (3.3)  C·CIP·G·K·RA  5  4              1 (20.0)  1 (0.8)  C·DOX·E·RA·TE  5  4            1 (12.5)    1 (0.8)  CAZ·CC·CIP·G·K  5  4              1 (20.0)  1 (0.8)  AM·CIP·DOX·P·TE  5  3  1 (3.1)      1 (8.3)        2 (1.7)  AM·G·K·P·TE  5  3    6 (18.2)            6 (5.0)  AM·CIP·DOX·P  4  3      1 (3.5)          1 (0.8)  AM·CIP·P·TE  4  3  1 (3.1)    1 (3.5)  1 (8.3)        3 (2.5)  AM·K·P·TE  4  3    4 (12.1)            4 (3.3)  Total (%)      8 (25.0)  17 (51.5)  5 (17.9)  6 (50.0)  1 (33.3)  1 (12.5)  5 (100.0)  43 (35.5)  AM, ampicillin; AmC, amoxicillin-clavulanic acid; C, chloramphenicol; CAZ, ceftazidime; CC, clindamycin; CF, cephalothin; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; CZ, cefazolin; DOX, doxycycline; E, erythromycin; FEP, cefepime; G, gentamicin; K, kanamycin; P, penicillin; RA, rifampin; SXT, sulfamethoxazole/trimethoprim; TE, tetracycline. View Large Mutations in gyrA and parC in CIP-resistant S. aureus The proportions of 41 CIP-resistant isolates with gyrA and/or parC mutations are shown in Table 4. The 5 different combinations of amino acid mutations in the QRDR are wild type/S80F, S84L/wild type, S84A/wild type, S84L/S80F, and S84L/S80F·E84K in gyrA/parC. Among these 5 combinations, the S84L/S80F mutation occurred in 31 (75.6%) isolates, for which the CIP and ENR MIC were in the range of 32 to 128 and 8 to 128 μg/mL, respectively. However, CIP and ENR showed MIC of 4 to 32 and 0.5 to 8 μg/mL, respectively, for isolates that had a single mutation in either gyrA or parC. Table 4. Amino acid changes in quinolone resistance determining region and corresponding minimum inhibitory concentrations of 41 ciprofloxacin-resistant Staphylococcus aureus from 7 integrated broiler chicken operations. Integrated broiler chicken operations  Amino acid change  MIC (μg/mL)  No. of isolates (%)  gyrA  parC  Ciprofloxacin  Enrofloxacin  A (n = 7)  Wta  S80F  8  2  1 (14.2)    S84L  S80F  32  32  1 (14.2)        64  8  1 (14.2)        128  8  4 (57.1)  B (n = 15)  Wt  S80F  4  0.5  1 (6.6)        32  32  1 (6.6)    S84L  S80F  32  8  3 (20.0)        32  16  1 (6.6)        32  32  1 (6.6)        64  8  4 (26.7)        64  32  2 (13.3)    S84L  S80F, E84K  32  16  1 (6.6)    S84L  Wt  32  1  1 (6.6)  C (n = 5)  S84L  S80F  32  8  2 (40.0)        64  8  2 (40.0)    S84L  Wt  64  8  1 (20.0)  D (n = 8)  Wt  S80F  4  1  1 (12.5)    S84L  S80F  32  8  2 (25.0)        32  32  1 (12.5)        64  8  2 (25.0)        64  32  1 (12.5)    S84A  Wt  16  4  1 (12.5)  E (n = 1)  S84L  S80F  64  128  1 (100.0)  F (n = 0)  -  -  -  -  -  G (n = 5)  S84L  S80F  4  8  3 (60.0)    S84L  Wt  4  8  2 (40.0)  Integrated broiler chicken operations  Amino acid change  MIC (μg/mL)  No. of isolates (%)  gyrA  parC  Ciprofloxacin  Enrofloxacin  A (n = 7)  Wta  S80F  8  2  1 (14.2)    S84L  S80F  32  32  1 (14.2)        64  8  1 (14.2)        128  8  4 (57.1)  B (n = 15)  Wt  S80F  4  0.5  1 (6.6)        32  32  1 (6.6)    S84L  S80F  32  8  3 (20.0)        32  16  1 (6.6)        32  32  1 (6.6)        64  8  4 (26.7)        64  32  2 (13.3)    S84L  S80F, E84K  32  16  1 (6.6)    S84L  Wt  32  1  1 (6.6)  C (n = 5)  S84L  S80F  32  8  2 (40.0)        64  8  2 (40.0)    S84L  Wt  64  8  1 (20.0)  D (n = 8)  Wt  S80F  4  1  1 (12.5)    S84L  S80F  32  8  2 (25.0)        32  32  1 (12.5)        64  8  2 (25.0)        64  32  1 (12.5)    S84A  Wt  16  4  1 (12.5)  E (n = 1)  S84L  S80F  64  128  1 (100.0)  F (n = 0)  -  -  -  -  -  G (n = 5)  S84L  S80F  4  8  3 (60.0)    S84L  Wt  4  8  2 (40.0)  aWild type; non-mutation. View Large Antimicrobial resistance of MRSA The characteristics of 4 MRSA isolates are shown in Table 5. All the MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, with OX, CIP, and ENR MIC ranges of 16 to 128, 32 to 64, and 8 to 128 μg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Table 5. Antimicrobial resistance of 4 methicillin-resistant Staphylococcus aureus isolated from 7 integrated broiler chicken operations. Integrated broiler chicken operations  Strains  Antimicrobial patterns  No. of antimicrobials  No. of classes  PVL gene  MIC (μg/mL)  Amino acid change  OX  CIP  ENR  gyrA  parC  B  113–2  AM·AmC·CAZ·CC·CF·CIP·  13  7  -  128  32  8  S84L  S80F      CTX·CXM·CZ·E·FEP·P·TE                  C  73–1  AM·CC·CIP·E·P·TE  6  5  -  128  32  8  S84L  S80F    75–1  AM·CC·CIP·E·P·TE  6  5  -  128  64  8  S84L  S80F  E  189–1  AM·AmC·CAZ·CC·CF·CIP·  14  7  -  16  64  128  S84L  S80F      CTX·CXM·CZ·DOX·E·FEP·P·TE                  Integrated broiler chicken operations  Strains  Antimicrobial patterns  No. of antimicrobials  No. of classes  PVL gene  MIC (μg/mL)  Amino acid change  OX  CIP  ENR  gyrA  parC  B  113–2  AM·AmC·CAZ·CC·CF·CIP·  13  7  -  128  32  8  S84L  S80F      CTX·CXM·CZ·E·FEP·P·TE                  C  73–1  AM·CC·CIP·E·P·TE  6  5  -  128  32  8  S84L  S80F    75–1  AM·CC·CIP·E·P·TE  6  5  -  128  64  8  S84L  S80F  E  189–1  AM·AmC·CAZ·CC·CF·CIP·  14  7  -  16  64  128  S84L  S80F      CTX·CXM·CZ·DOX·E·FEP·P·TE                  AM, ampicillin; AmC, amoxicillin-clavulanic acid; CAZ, ceftazidime; CC, clindamycin; CF, cephalothin; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; CZ, cefazolin; DOX, doxycycline; E, erythromycin; ENR, enrofloxacin; FEP, cefepime; OX, oxacillin; P, penicillin; TE, tetracycline. View Large DISCUSSION Many foods, particularly those of animal origin, have been identified as vehicles for the transmission of pathogens to human beings being spread through the processing and kitchen environments (Frye and Fedorka-Cray, 2007). In particular, foods originating from poultry are important sources of foodborne illnesses in humans (Kitai et al., 2005; Jackson et al., 2013; Kim et al., 2016). The poultry industry is a vertically integrated production, processing, and distribution system. Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The present study indicates that the prevalence of S. aureus in chicken meats from 7 different integrated practices during 2016 was 47.0%. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among operations. Although there was a difference in the numbers of sample sizes, the prevalence of S. aureus might be associated with differences in the hygiene and sanitation levels of each operation. In previous studies, the frequency of S. aureus in chicken meat was found to be 30.4% in Korea, 38% in Poland, and 17.8% in the United States (Woo, 2007; Hanson et al., 2011; Krupa et al., 2014). The number of S. aureus isolates obtained in this study was higher than in the other countries, and this difference also may have resulted from the production techniques used, as well as in personal hygiene, slaughterhouse hygiene, and other practices through to the food chain. In this study, antimicrobial susceptibility testing was performed on a total of 121 S. aureus isolates, and the percentage of the antimicrobial resistance varies from company to company. When comparing the antimicrobial resistance by operation system, 25.0 to 100% of the isolates had resistance to P, 12.5 to 60.0% to TE, 0 to 100% to CIP, 9.1 to 60.0% to DOX, and 0 to 33.3% to E. Antimicrobial resistance occurs when bacteria change in response to the use of these antimicrobial drugs. Vertical integration has allowed for the strict maintenance of biosecurity measures, vaccine programs, and antibiotic applications. Therefore, our results suggest that the tendency toward antimicrobial resistance occurs for the antibiotics routinely used for growth promotion, disease prevention, or therapeutic purposes in each operation system. Our results were comparable to data from the Korea Quality Improvement Authority report (QIA, 2015), in which S. aureus from domestic chicken meat showed resistance to P (50.0%), TE (39.3%), and CIP (39.3%). However, the QIA (2015) also reported that S. aureus from imported chicken meat from Brazil, the United States, and Denmark showed different degrees of resistance to P (100.0%), TE (33.3%), and CIP (0.0%). The difference in antimicrobial resistance between the meats from Korea and those from other countries also may be associated with the usage of antibiotics in each country. Fluoroquinolones are antibiotics classified by the World Health Organization as “critically important in human medicine” owing to their importance for treating infections from pathogens such as Campylobacter, Salmonella, and E. coli (WHO, 2015). Mass medication of poultry with fluoroquinolones is still permitted in Korea. Fluoroquinolone resistance in S. aureus has mainly been attributed to mutations occurring in the QRDR of parC (encoding topoisomerase IV) and of gyrA (encoding DNA gyrase A) (Kwak et al., 2013). In our study, 75.6% of CIP-resistant isolates showed a double amino-acid exchange in both gyrA and parC with CIP MIC of ≥32 μg/mL. The extensive use of fluoroquinolones has led to the emergence of fluoroquinolone-resistant S. aureus, in which such double mutants have demonstrated a particularly high level of resistance to fluoroquinolones (Hashem et al., 2013). The co-occurrence of resistance to a series of antibiotics also was shown in this study. Petrelli et al. (2008) reported a significant correlation between OX resistance and resistance to E, CC, G, and CIP. Karam et al. (2016) reported that extended-spectrum beta-lactamase (ESBL)-producing pathogens are often resistant to fluoroquinolones and aminoglycosides, since the resistance mechanisms for these classes of antibiotics are carried on the same large plasmids that contain the genetic elements for ESBL production. In this study, we found 4 MRSA isolates from 3 of the 7 integrated broiler operations. These isolates also exhibited co-resistance toward more than 5 classes of antibiotics and were significantly associated with resistance to CIP. The prevalence of MRSA in chicken meat is 1.2% in the United States (Abdalrahman et al., 2015) and 4% in Saudi Arabia (Raji et al., 2016). In Korea, Kim et al. (2015) reported the prevalence of MRSA in domestic chicken meat to be 0% between 2009 and 2011. However, in our current study, 4 MRSA isolates among 121 S. aureus isolates were obtained from chicken meat in 2016, although the prevalence may be linked to differences in the integrated broiler operation systems studied. To our best knowledge, this study is the first to investigate the characteristics of S. aureus isolates from different integrated chicken operations in Korea. Our findings suggest that S. aureus with resistance to important antimicrobial compounds can now be found in association with integrated broiler operations, providing the data to support the development of a monitoring and prevention program in integrated operations. ACKNOWLEDGEMENTS This work was supported by a fund (Z-1543081–2016-17–01) by the Research of Animal and Plant Quarantine Agency, South Korea. REFERENCES Abdalrahman L. S., Stanley A., Wells H., Fakhr M. K.. 2015. Isolation, virulence, and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA) strains from Oklahoma retail poultry meats. Int. J. Environ. Res. Health.  12: 6148– 6161. Google Scholar CrossRef Search ADS   Alliance for the Prudent Use of Antibiotics (APUA). 2010. Policy brief and recommendations #4 misuse of antibiotics in food animal production antibiotic misuse in food animals – Time for change . Boston, MA. 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Characteristics of the antimicrobial resistance of Staphylococcus aureus isolated from chicken meat produced by different integrated broiler operations in Korea

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pex357
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Abstract

Abstract Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The purpose of this study was to investigate the prevalence of Staphylococcus aureus (S. aureus) and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems in Korea. Among 200 chicken meat samples, 94 were observed to be positive for S. aureus. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among the operations. Four methicillin-resistant S. aureus isolates (MRSA) were recovered from 3 different operations. A high proportion of the S. aureus isolates were resistant to penicillins (51.2%), tetracycline (38.8%), and ciprofloxacin (CIP; 33.9%). Especially, 3 different operations showed a high number of CIP resistance (45.5∼100%) and multidrug resistance (50.0∼100%). Among 41 CIP-resistant S. aureus isolates, 75.6% showed a double amino-acid exchange of both gyrA and parC, with CIP minimum inhibitory concentrations (MIC) of ≥32 μg/mL. Four MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, exhibiting oxacillin, CIP, and enrofloxacin MIC ranges of 16 to 128, 32 to 64, and 8 to 128 μg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Our findings suggest that S. aureus with resistance to important antimicrobial compounds can now be found in association with integrated broiler operations, providing the data to support the development of a monitoring and prevention program in integrated operations. INTRODUCTION Staphylococcus aureus (S. aureus) is one of the most common causes of foodborne diseases in the world and produces gastrointestinal illness through a wide variety of toxins, including staphylococcal enterotoxins. In Korea, S. aureus was the fourth most frequent pathogen, after pathogenic Escherichia coli, Salmonella, and Vibrio spp. (Hyeon et al., 2013). The Korea Food and Drug Administration reported that a total of 1,472 cases were reported from 38 staphylococcal food poisoning outbreaks from 2012 to 2016 in Korea. (http://foodsafetykorea.go.kr/portal/healthyfoodlife/foodPoisoningStat.do?menu_grp=MENU_NEW02&menu_no=2786). S. aureus is a common commensal of the skin and mucosal membranes of humans, with estimates of 20 to 30% for persistent and 60% for intermittent colonizations (Kluytmans and Wertheim, 2005). However, S. aureus of animal origin may be transmitted to humans through direct contact with animals or via exposure to contaminated food (Lowder et al., 2009). Epidemiologically, poultry meat is of paramount importance and is still inculpated as a primary source of human food poisoning (Abdalrahman et al., 2015; Owuna et al., 2015; Bortolaia et al., 2016). Zoonotic transmission of S. aureus from poultry has usually been studied in relation to antimicrobial resistance. It is considered to be a significantly disturbing issue in the poultry industry owing to its impact on public health, and is a challenge to medical and veterinary officials worldwide (APUA, 2010; Ruban and Fairoze, 2011). Especially, methicillin-resistant S. aureus (MRSA) has been isolated in chicken meats (Lim et at., 2010; Velasco et al., 2015) and is considered a source of human infections caused by consuming contaminated food products made from animals (Lee, 2003). The Korea Animal Health Products Association reported that around 1,500 tons of antimicrobials were sold each year during 2003 to 2007, but less than 1,000 tons were sold for the 4 consecutive years from 2011 to 2015. Although tetracyclines and penicillins were the largest selling antimicrobials, the sales of most antimicrobials have gradually decreased. However, the sales of phenicols and cephalosporins have increased by 3 and 5 times from 2007 to 2015, respectively (QIA, 2015). Presently, the broiler chicken industry operates largely through vertical integration, with company ownership of breeding farms, multiplication farms, hatcheries, feed mills, some broiler growing farms, and processing plants. In Korea, several large integrated companies supply about 80% of the broiler chickens on the market, (KAPE, 2015). The livestock on the integrated farms, which includes chicken, is reared intensively, and antimicrobial agents are used as growth promoters and for prophylactic and therapeutic treatments. The objective of this study was to investigate the prevalence of S. aureus and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems. MATERIALS AND METHODS Sample collection A total of 200 chicken meats was collected at 4 retail markets, visiting 3 or more times during the year 2016. These meats were produced by 50 broiler farms and divided into 7 different integrated broiler operations supplying about 80% of the broiler chickens in Korea. Four meats from the same farm origin were sampled and tested for this study. Each meat was aseptically placed into a vacuum bag (Sealed Air, Elmwood Park, NJ), and 400mL of sterile buffered peptone water (BD Biosciences, Sparks, MD) were added. The bag was shaken 50 times, and approximately 50mL of the rinse water were transferred into a sterile specimen cup. Bacterial isolation Microbiological analyses were performed according to the Processing and Ingredients Specification of Livestock Products published by the Ministry of Food and Drug Safety (KFDS, 2014). In brief, 25 mL of meat rinse water were added to 225 mL of tryptic soy broth (TSB; BD Biosciences) containing 10% NaCl and incubated at 37°C for 20 to 24 hours. After enrichment, the bacteria-containing TSB was streaked onto Baird-Parker agar (Oxoid Ltd., Hampshire, UK) with egg yolk tellurite emulsion (Oxoid) and incubated at 37°C for 48 hours. Two typical colonies of S. aureus from one meat origin were picked and identified by standard microbiological culture-based tests, including Gram staining; catalase (using 3% hydrogen peroxide), indole, oxidase, coagulase, citrate, urease, Voges-Proskauer, and DNase testing; and tests for sugar fermentation and mannitol salt agar oxidation and fermentation (Cruickshank et al., 1975). PCR identification of S. aureus was carried out as described by Manson et al. (2001). If 2 isolates from the same origin showed the same antimicrobial susceptibility patterns, then only one isolate was randomly chosen and included in this study. Antimicrobial susceptibility tests All S. aureus isolates were investigated for their antimicrobial resistance with the disc diffusion test, using the following antibiotic discs (BD): amikacin (AN, 30 μg), amoxicillin/clavulanic acid (AmC, 20/10 μg), ampicillin (10 μg), cefazolin (30 μg), cefepime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CC, 2 μg), doxycycline (DOX, 30 μg), erythromycin (E, 15 μg), gentamicin (G, 10 μg), kanamycin (30 μg), penicillin (P, 10 μg), rifampin (5 μg), sulfamethoxazole/trimethoprim (23.75/1.25 μg), tetracycline (TE, 30 μg) and vancomycin (VA, 30 μg), and nitrofurantoin (F/M, 300 μg) and teicoplanin (TEC, 30 μg) purchased from Oxoid. Results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2013). Multidrug resistance (MDR) was defined as acquired non-susceptibility to at least one agent in 3 or more antimicrobial categories. Minimum inhibitory concentrations The minimum inhibitory concentrations (MIC) were determined by standard agar dilution methods with Mueller–Hinton agar (BD Biosciences), according to the recommendations of the CLSI (2013). The antimicrobial agents used in this study were CIP, enrofloxacin (ENR), and oxacillin (OX) at concentrations ranging from 0.125 to 256 μg/mL. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The breakpoint was determined using the S. aureus criteria set by the CLSI (2013). S. aureus ATCC 29,213 was used as a quality control, as recommended by the CLSI. Screening for mutations in gyrA and parC For CIP-resistant strains, PCR amplification of the quinolone resistance-determining region (QRDR) of gyrA and parC was carried out using the primers: gyrA-F (5΄-AATGAACAAGGTATGACACC-3΄) and gyrA-R (5΄-TACGCGCTTCAGTATAACGC-3΄); and parC-F (5΄-ACTTGAAGATGTTTTAGGTGAT-3΄) and parC-R (5΄-TTAGGAAATCTTGATGGCAA-3΄) (Kwak et al., 2013). The amplified DNA was purified using a PCR purification kit (Qiagen, Valencia, CA) and sequenced (Cosmogen Tech, Daejoen, Korea). The DNA sequences were compared with those of the standard gyrA (GenBank Accession No. ABD29197.1) and parC (GenBank Accession No. ABD30448.1) genes. Detection of mecA and pvl The mecA and pvl genes were detected by PCR using the primers as described previously (Lina et al., 1999; Oliveira and Lencastre, 2002) RESULTS Prevalence of S. aureus The prevalence of S. aureus in the chicken meats collected from a retail market is shown in Table 1. Among 200 chicken meats, 94 were observed to be positive for S. aureus. Among chicken meats that had originated from 7 integrated broiler chicken operations, those from operation C had the highest prevalence of S. aureus (59.4%, 19 of 32 samples), and operation E showed the lowest prevalence (25.0%, 3 of 12 samples). Four MRSA isolates were obtained from operations B (one isolate), C (2 isolates), and E (one isolate). However, a higher number of CIP-resistant S. aureus isolates were obtained from operations B (45.5%, 15 of 33 isolates), D (66.7%, 8 of 12 isolates), and G (100%, 5 of 5 isolates). Table 1. Prevalence of Staphylococcus aureus from 7 integrated broiler chicken operations.   Integrated broiler chicken operations  A  B  C  D  E  F  G  Total (%)  No. of farms  14  11  8  8  3  3  3  50  No. of chicken meat tested  56  44  32  32  12  12  12  200  No. of chicken meat isolated Staphylococcus aureus (%)  28 (53.8)  22 (50.0)  19 (59.4)  11 (34.4)  3 (25.0)  7 (58.3)  4 (33.3)  94 (47.0)  No. of Staphylococcus aureus isolatesa  32  33  28  12  3  8  5  121  No. of Methicillin- resistant Staphylococcus aureus (%)  0 (0.0)  1 (3.0)  2 (7.1)  0 (0.0)  1(33.3)  0 (0.0)  0 (0.0)  4 (3.3)  No. of Ciprofloxacin- resistant Staphylococcus aureus (%)  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)    Integrated broiler chicken operations  A  B  C  D  E  F  G  Total (%)  No. of farms  14  11  8  8  3  3  3  50  No. of chicken meat tested  56  44  32  32  12  12  12  200  No. of chicken meat isolated Staphylococcus aureus (%)  28 (53.8)  22 (50.0)  19 (59.4)  11 (34.4)  3 (25.0)  7 (58.3)  4 (33.3)  94 (47.0)  No. of Staphylococcus aureus isolatesa  32  33  28  12  3  8  5  121  No. of Methicillin- resistant Staphylococcus aureus (%)  0 (0.0)  1 (3.0)  2 (7.1)  0 (0.0)  1(33.3)  0 (0.0)  0 (0.0)  4 (3.3)  No. of Ciprofloxacin- resistant Staphylococcus aureus (%)  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  aIf two isolates from the same origin showed the same antimicrobial susceptibility patterns, only one isolate was included. View Large Antimicrobial resistance profile The results of the antimicrobial resistance analysis of the S. aureus isolates are shown in Table 2. A high proportion of S. aureus isolates was resistant to penicillins (62 isolates, 51.2%), TE (47 isolates, 38.8%), CIP (41 isolates, 33.9%), K (30 isolates, 24.7%), DOX (25 isolates, 20.7%), and E (25 isolates, 20.7%). Although isolates from operations C and F showed only 25.0 and 12.5% resistance to penicillins, respectively, the isolates from the other integrated operations were the most frequently resistant to this antibiotic. Moreover, isolates from operation B showed the highest resistance to K (48.5%) and TE (48.5%), those from operation D had the highest resistance to TE (58.3%) and CIP (66.7%), and isolates from operation G were the most resistant to AmC (60.0%), TE (60.0%), DOX (60.0%), and CIP (100%). Table 2. Distribution of antimicrobial resistance of 121 Staphylococcus aureus from 7 integrated broiler chicken operations.   Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  PNICILLINS  Penicillin  25 (78.1)a  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  Ampicillin  25 (78.1)  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS  Amoxicillin/Clavulanic acid  1 (3.1)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  3 (60.0)  6 (5.0)  CEPHEMS (PARENTERAL)  Cefazolin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cephalothin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefuroxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefotaxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Ceftazidime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  1 (20.0)  3 (2.5)  Cefepime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  AMIOGLYCOSIDES  Gentamicin  1 (3.1)  12 (36.4)  8 (28.6)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  23 (19.0)  Kanamycin  2 (6.3)  16 (48.5)  10 (35.7)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  30 (24.7)  MARCROLIDES  Erythromycin  6 (18.8)  9 (27.3)  4 (14.3)  4 (33.3)  1 (33.3)  1 (12.5)  0 (0.0)  25 (20.7)  TETEACYCLINES  Tetracycline  7 (21.9)  16 (48.5)  12 (42.9)  7 (58.3)  1 (33.3)  1 (12.5)  3 (60.0)  47 (38.8)  Doxycycline  6 (18.8)  3 (9.1)  8 (28.6)  3 (25.0)  1 (33.3)  1 (12.5)  3 (60.0)  25 (20.7)  FLUOROQUINOLONES  Ciprofloxacin  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  LINCOSAMIDES  Clindamycin  6 (18.8)  9 (27.3)  3 (10.7)  4 (33.3)  1 (33.3)  0 (0.0)  0 (0.0)  23 (19.0)  FOLATE PATHWAY INHIBITORS  Sulfamethoxazole/Trimethoprim  1 (3.1)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (0.8)  PHENICOLS  Chloramphenicol  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (12.5)  2 (40.0)  3 (2.5)  ANSAMYCINS  Rifampin  0 (0.0)  0 (0.0)  1 (3.5)  0 (0.0)  0 (0.0)  1 (12.5)  1 (20.0)  3 (2.5)    Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  PNICILLINS  Penicillin  25 (78.1)a  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  Ampicillin  25 (78.1)  17 (51.5)  7 (25.0)  6 (50.0)  3 (100.0)  1 (12.5)  3 (60.0)  62 (51.2)  β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS  Amoxicillin/Clavulanic acid  1 (3.1)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  3 (60.0)  6 (5.0)  CEPHEMS (PARENTERAL)  Cefazolin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cephalothin  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefuroxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Cefotaxime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  Ceftazidime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  1 (20.0)  3 (2.5)  Cefepime  0 (0.0)  1 (3.0)  0 (0.0)  0 (0.0)  1 (33.3)  0 (0.0)  0 (0.0)  2 (1.7)  AMIOGLYCOSIDES  Gentamicin  1 (3.1)  12 (36.4)  8 (28.6)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  23 (19.0)  Kanamycin  2 (6.3)  16 (48.5)  10 (35.7)  0 (0.0)  0 (0.0)  0 (0.0)  2 (40.0)  30 (24.7)  MARCROLIDES  Erythromycin  6 (18.8)  9 (27.3)  4 (14.3)  4 (33.3)  1 (33.3)  1 (12.5)  0 (0.0)  25 (20.7)  TETEACYCLINES  Tetracycline  7 (21.9)  16 (48.5)  12 (42.9)  7 (58.3)  1 (33.3)  1 (12.5)  3 (60.0)  47 (38.8)  Doxycycline  6 (18.8)  3 (9.1)  8 (28.6)  3 (25.0)  1 (33.3)  1 (12.5)  3 (60.0)  25 (20.7)  FLUOROQUINOLONES  Ciprofloxacin  7 (21.9)  15 (45.5)  5 (17.9)  8 (66.7)  1 (33.3)  0 (0.0)  5 (100.0)  41 (33.9)  LINCOSAMIDES  Clindamycin  6 (18.8)  9 (27.3)  3 (10.7)  4 (33.3)  1 (33.3)  0 (0.0)  0 (0.0)  23 (19.0)  FOLATE PATHWAY INHIBITORS  Sulfamethoxazole/Trimethoprim  1 (3.1)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (0.8)  PHENICOLS  Chloramphenicol  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  0 (0.0)  1 (12.5)  2 (40.0)  3 (2.5)  ANSAMYCINS  Rifampin  0 (0.0)  0 (0.0)  1 (3.5)  0 (0.0)  0 (0.0)  1 (12.5)  1 (20.0)  3 (2.5)  aNo. of isolates shown resistance (%). All isolates showed sensitivity to kanamycin, nitrofurantoin, vancomycin, and teicoplanin tested in this study. View Large The distribution of MDR is shown in Table 3. Forty-three of 121 isolates showed resistance from 3 to 7 classes of antimicrobial agents. Operations B (51.5%, 17 of 33 isolates), D (50.0%, 6 of 12 isolates), and G (100%, 5 of 5 isolates), in particular, showed a high proportion of isolates with MDR. Table 3. Multidrug resistance patterns among 121 Staphylococcus aureus from 7 integrated broiler chicken operations. Antimicrobial resistance patterns  No. of antimicrobials  No. of classes  Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  AM·AmC·CAZ·CC·CF·CIP·  14  7          1 (33.3)      1 (0.8)  CTX·CXM·CZ·DOX·E·FEP·P·TE                      AM·AmC·CAZ·CC·CF·CIP·  13  7    1 (3.0)            1 (0.8)  CTX·CXM·CZ·E·FEP·P·TE                      AM·C·CC·CIP·E·P·K·SXT  8  7  1 (3.1)              1 (0.8)  AM·C·CC·CIP·CTX·E·P·TE  8  6  2 (6.3)              2 (1.7)  AM·CC·CIP·DOX·E·P·TE  7  5  3 (9.4)  2 (6.1)            5 (4.1)  AM·CC·E·G·K·P·TE  7  5    1 (3.0)            1 (0.8)  AM·CC·CIP·E·P·TE  6  5    1 (3.0)  3 (10.7)  2 (16.7)        6 (5.0)  AM·AmC·CIP·DOX·P·TE  6  4              3 (60.0)  3 (2.5)  AM·CC·CIP·E·P  5  4    2 (6.1)    2 (16.7)        4 (3.3)  C·CIP·G·K·RA  5  4              1 (20.0)  1 (0.8)  C·DOX·E·RA·TE  5  4            1 (12.5)    1 (0.8)  CAZ·CC·CIP·G·K  5  4              1 (20.0)  1 (0.8)  AM·CIP·DOX·P·TE  5  3  1 (3.1)      1 (8.3)        2 (1.7)  AM·G·K·P·TE  5  3    6 (18.2)            6 (5.0)  AM·CIP·DOX·P  4  3      1 (3.5)          1 (0.8)  AM·CIP·P·TE  4  3  1 (3.1)    1 (3.5)  1 (8.3)        3 (2.5)  AM·K·P·TE  4  3    4 (12.1)            4 (3.3)  Total (%)      8 (25.0)  17 (51.5)  5 (17.9)  6 (50.0)  1 (33.3)  1 (12.5)  5 (100.0)  43 (35.5)  Antimicrobial resistance patterns  No. of antimicrobials  No. of classes  Integrated broiler chicken operations  A (n = 32)  B (n = 33)  C (n = 28)  D (n = 12)  E (n = 3)  F (n = 8)  G (n = 5)  Total (n = 121)  AM·AmC·CAZ·CC·CF·CIP·  14  7          1 (33.3)      1 (0.8)  CTX·CXM·CZ·DOX·E·FEP·P·TE                      AM·AmC·CAZ·CC·CF·CIP·  13  7    1 (3.0)            1 (0.8)  CTX·CXM·CZ·E·FEP·P·TE                      AM·C·CC·CIP·E·P·K·SXT  8  7  1 (3.1)              1 (0.8)  AM·C·CC·CIP·CTX·E·P·TE  8  6  2 (6.3)              2 (1.7)  AM·CC·CIP·DOX·E·P·TE  7  5  3 (9.4)  2 (6.1)            5 (4.1)  AM·CC·E·G·K·P·TE  7  5    1 (3.0)            1 (0.8)  AM·CC·CIP·E·P·TE  6  5    1 (3.0)  3 (10.7)  2 (16.7)        6 (5.0)  AM·AmC·CIP·DOX·P·TE  6  4              3 (60.0)  3 (2.5)  AM·CC·CIP·E·P  5  4    2 (6.1)    2 (16.7)        4 (3.3)  C·CIP·G·K·RA  5  4              1 (20.0)  1 (0.8)  C·DOX·E·RA·TE  5  4            1 (12.5)    1 (0.8)  CAZ·CC·CIP·G·K  5  4              1 (20.0)  1 (0.8)  AM·CIP·DOX·P·TE  5  3  1 (3.1)      1 (8.3)        2 (1.7)  AM·G·K·P·TE  5  3    6 (18.2)            6 (5.0)  AM·CIP·DOX·P  4  3      1 (3.5)          1 (0.8)  AM·CIP·P·TE  4  3  1 (3.1)    1 (3.5)  1 (8.3)        3 (2.5)  AM·K·P·TE  4  3    4 (12.1)            4 (3.3)  Total (%)      8 (25.0)  17 (51.5)  5 (17.9)  6 (50.0)  1 (33.3)  1 (12.5)  5 (100.0)  43 (35.5)  AM, ampicillin; AmC, amoxicillin-clavulanic acid; C, chloramphenicol; CAZ, ceftazidime; CC, clindamycin; CF, cephalothin; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; CZ, cefazolin; DOX, doxycycline; E, erythromycin; FEP, cefepime; G, gentamicin; K, kanamycin; P, penicillin; RA, rifampin; SXT, sulfamethoxazole/trimethoprim; TE, tetracycline. View Large Mutations in gyrA and parC in CIP-resistant S. aureus The proportions of 41 CIP-resistant isolates with gyrA and/or parC mutations are shown in Table 4. The 5 different combinations of amino acid mutations in the QRDR are wild type/S80F, S84L/wild type, S84A/wild type, S84L/S80F, and S84L/S80F·E84K in gyrA/parC. Among these 5 combinations, the S84L/S80F mutation occurred in 31 (75.6%) isolates, for which the CIP and ENR MIC were in the range of 32 to 128 and 8 to 128 μg/mL, respectively. However, CIP and ENR showed MIC of 4 to 32 and 0.5 to 8 μg/mL, respectively, for isolates that had a single mutation in either gyrA or parC. Table 4. Amino acid changes in quinolone resistance determining region and corresponding minimum inhibitory concentrations of 41 ciprofloxacin-resistant Staphylococcus aureus from 7 integrated broiler chicken operations. Integrated broiler chicken operations  Amino acid change  MIC (μg/mL)  No. of isolates (%)  gyrA  parC  Ciprofloxacin  Enrofloxacin  A (n = 7)  Wta  S80F  8  2  1 (14.2)    S84L  S80F  32  32  1 (14.2)        64  8  1 (14.2)        128  8  4 (57.1)  B (n = 15)  Wt  S80F  4  0.5  1 (6.6)        32  32  1 (6.6)    S84L  S80F  32  8  3 (20.0)        32  16  1 (6.6)        32  32  1 (6.6)        64  8  4 (26.7)        64  32  2 (13.3)    S84L  S80F, E84K  32  16  1 (6.6)    S84L  Wt  32  1  1 (6.6)  C (n = 5)  S84L  S80F  32  8  2 (40.0)        64  8  2 (40.0)    S84L  Wt  64  8  1 (20.0)  D (n = 8)  Wt  S80F  4  1  1 (12.5)    S84L  S80F  32  8  2 (25.0)        32  32  1 (12.5)        64  8  2 (25.0)        64  32  1 (12.5)    S84A  Wt  16  4  1 (12.5)  E (n = 1)  S84L  S80F  64  128  1 (100.0)  F (n = 0)  -  -  -  -  -  G (n = 5)  S84L  S80F  4  8  3 (60.0)    S84L  Wt  4  8  2 (40.0)  Integrated broiler chicken operations  Amino acid change  MIC (μg/mL)  No. of isolates (%)  gyrA  parC  Ciprofloxacin  Enrofloxacin  A (n = 7)  Wta  S80F  8  2  1 (14.2)    S84L  S80F  32  32  1 (14.2)        64  8  1 (14.2)        128  8  4 (57.1)  B (n = 15)  Wt  S80F  4  0.5  1 (6.6)        32  32  1 (6.6)    S84L  S80F  32  8  3 (20.0)        32  16  1 (6.6)        32  32  1 (6.6)        64  8  4 (26.7)        64  32  2 (13.3)    S84L  S80F, E84K  32  16  1 (6.6)    S84L  Wt  32  1  1 (6.6)  C (n = 5)  S84L  S80F  32  8  2 (40.0)        64  8  2 (40.0)    S84L  Wt  64  8  1 (20.0)  D (n = 8)  Wt  S80F  4  1  1 (12.5)    S84L  S80F  32  8  2 (25.0)        32  32  1 (12.5)        64  8  2 (25.0)        64  32  1 (12.5)    S84A  Wt  16  4  1 (12.5)  E (n = 1)  S84L  S80F  64  128  1 (100.0)  F (n = 0)  -  -  -  -  -  G (n = 5)  S84L  S80F  4  8  3 (60.0)    S84L  Wt  4  8  2 (40.0)  aWild type; non-mutation. View Large Antimicrobial resistance of MRSA The characteristics of 4 MRSA isolates are shown in Table 5. All the MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, with OX, CIP, and ENR MIC ranges of 16 to 128, 32 to 64, and 8 to 128 μg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Table 5. Antimicrobial resistance of 4 methicillin-resistant Staphylococcus aureus isolated from 7 integrated broiler chicken operations. Integrated broiler chicken operations  Strains  Antimicrobial patterns  No. of antimicrobials  No. of classes  PVL gene  MIC (μg/mL)  Amino acid change  OX  CIP  ENR  gyrA  parC  B  113–2  AM·AmC·CAZ·CC·CF·CIP·  13  7  -  128  32  8  S84L  S80F      CTX·CXM·CZ·E·FEP·P·TE                  C  73–1  AM·CC·CIP·E·P·TE  6  5  -  128  32  8  S84L  S80F    75–1  AM·CC·CIP·E·P·TE  6  5  -  128  64  8  S84L  S80F  E  189–1  AM·AmC·CAZ·CC·CF·CIP·  14  7  -  16  64  128  S84L  S80F      CTX·CXM·CZ·DOX·E·FEP·P·TE                  Integrated broiler chicken operations  Strains  Antimicrobial patterns  No. of antimicrobials  No. of classes  PVL gene  MIC (μg/mL)  Amino acid change  OX  CIP  ENR  gyrA  parC  B  113–2  AM·AmC·CAZ·CC·CF·CIP·  13  7  -  128  32  8  S84L  S80F      CTX·CXM·CZ·E·FEP·P·TE                  C  73–1  AM·CC·CIP·E·P·TE  6  5  -  128  32  8  S84L  S80F    75–1  AM·CC·CIP·E·P·TE  6  5  -  128  64  8  S84L  S80F  E  189–1  AM·AmC·CAZ·CC·CF·CIP·  14  7  -  16  64  128  S84L  S80F      CTX·CXM·CZ·DOX·E·FEP·P·TE                  AM, ampicillin; AmC, amoxicillin-clavulanic acid; CAZ, ceftazidime; CC, clindamycin; CF, cephalothin; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; CZ, cefazolin; DOX, doxycycline; E, erythromycin; ENR, enrofloxacin; FEP, cefepime; OX, oxacillin; P, penicillin; TE, tetracycline. View Large DISCUSSION Many foods, particularly those of animal origin, have been identified as vehicles for the transmission of pathogens to human beings being spread through the processing and kitchen environments (Frye and Fedorka-Cray, 2007). In particular, foods originating from poultry are important sources of foodborne illnesses in humans (Kitai et al., 2005; Jackson et al., 2013; Kim et al., 2016). The poultry industry is a vertically integrated production, processing, and distribution system. Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The present study indicates that the prevalence of S. aureus in chicken meats from 7 different integrated practices during 2016 was 47.0%. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among operations. Although there was a difference in the numbers of sample sizes, the prevalence of S. aureus might be associated with differences in the hygiene and sanitation levels of each operation. In previous studies, the frequency of S. aureus in chicken meat was found to be 30.4% in Korea, 38% in Poland, and 17.8% in the United States (Woo, 2007; Hanson et al., 2011; Krupa et al., 2014). The number of S. aureus isolates obtained in this study was higher than in the other countries, and this difference also may have resulted from the production techniques used, as well as in personal hygiene, slaughterhouse hygiene, and other practices through to the food chain. In this study, antimicrobial susceptibility testing was performed on a total of 121 S. aureus isolates, and the percentage of the antimicrobial resistance varies from company to company. When comparing the antimicrobial resistance by operation system, 25.0 to 100% of the isolates had resistance to P, 12.5 to 60.0% to TE, 0 to 100% to CIP, 9.1 to 60.0% to DOX, and 0 to 33.3% to E. Antimicrobial resistance occurs when bacteria change in response to the use of these antimicrobial drugs. Vertical integration has allowed for the strict maintenance of biosecurity measures, vaccine programs, and antibiotic applications. Therefore, our results suggest that the tendency toward antimicrobial resistance occurs for the antibiotics routinely used for growth promotion, disease prevention, or therapeutic purposes in each operation system. Our results were comparable to data from the Korea Quality Improvement Authority report (QIA, 2015), in which S. aureus from domestic chicken meat showed resistance to P (50.0%), TE (39.3%), and CIP (39.3%). However, the QIA (2015) also reported that S. aureus from imported chicken meat from Brazil, the United States, and Denmark showed different degrees of resistance to P (100.0%), TE (33.3%), and CIP (0.0%). The difference in antimicrobial resistance between the meats from Korea and those from other countries also may be associated with the usage of antibiotics in each country. Fluoroquinolones are antibiotics classified by the World Health Organization as “critically important in human medicine” owing to their importance for treating infections from pathogens such as Campylobacter, Salmonella, and E. coli (WHO, 2015). Mass medication of poultry with fluoroquinolones is still permitted in Korea. Fluoroquinolone resistance in S. aureus has mainly been attributed to mutations occurring in the QRDR of parC (encoding topoisomerase IV) and of gyrA (encoding DNA gyrase A) (Kwak et al., 2013). In our study, 75.6% of CIP-resistant isolates showed a double amino-acid exchange in both gyrA and parC with CIP MIC of ≥32 μg/mL. The extensive use of fluoroquinolones has led to the emergence of fluoroquinolone-resistant S. aureus, in which such double mutants have demonstrated a particularly high level of resistance to fluoroquinolones (Hashem et al., 2013). The co-occurrence of resistance to a series of antibiotics also was shown in this study. Petrelli et al. (2008) reported a significant correlation between OX resistance and resistance to E, CC, G, and CIP. Karam et al. (2016) reported that extended-spectrum beta-lactamase (ESBL)-producing pathogens are often resistant to fluoroquinolones and aminoglycosides, since the resistance mechanisms for these classes of antibiotics are carried on the same large plasmids that contain the genetic elements for ESBL production. In this study, we found 4 MRSA isolates from 3 of the 7 integrated broiler operations. These isolates also exhibited co-resistance toward more than 5 classes of antibiotics and were significantly associated with resistance to CIP. The prevalence of MRSA in chicken meat is 1.2% in the United States (Abdalrahman et al., 2015) and 4% in Saudi Arabia (Raji et al., 2016). In Korea, Kim et al. (2015) reported the prevalence of MRSA in domestic chicken meat to be 0% between 2009 and 2011. However, in our current study, 4 MRSA isolates among 121 S. aureus isolates were obtained from chicken meat in 2016, although the prevalence may be linked to differences in the integrated broiler operation systems studied. To our best knowledge, this study is the first to investigate the characteristics of S. aureus isolates from different integrated chicken operations in Korea. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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